1. Field of the Invention
The present invention relates to an assay method and a microfluidic device for analyzing a subjects' body fluids containing leucocytes to determine if the subject has been previously exposed to the antigen. Specifically, the invention relates to an assay for observation, qualification and quantification of leucocyte measurement factors in a leucocyte containing fluid sample from a subject by exposure to a predetermined antigen in a novel microfluidic device in order to determine if there has been a prior exposure to the antigen.
2. Description of the Related Art
In leucocyte containing animals such as bird, mammals and especially humans, it is often necessary or determine if they have been previously exposed to a predetermined antigen. Antigens can be disease pathogens, allergy agents, toxins, infectious agents and the like. These days, because of the threat of bio-terrorism, not only are physicians concerned with normal exposure of their patients to various pathogens, the possibility of a civilian or military population being exposed to a terrorist act such as the anthrax events of late has created a need for tests which are simple, can be performed almost anywhere, are accurate and fast to determine if there has been a first prior exposure to a predetermined antigen in order to determine what if any method of treatment to use on the subject. In addition to the above, prior exposure to an antigen necessarily results after vaccination for infectious diseases. It is often desirable for a physician to determine that a vaccination has been successful in immunizing a subject or that after an extended time period that immunization from a subjects' prior vaccination remains above an acceptable level.
Antibodies are produced by certain lymphocytes, the B-lymphocytes, and are one of the body's defense mechanism against antigens in the blood stream and other body fluids containing leucocyte. The presence of circulating antibodies is an indicator that the subject is or has been actively fighting against such infectious agents as viral or bacterial antigens. A plethora of tests have been developed to directly and indirectly measure circulating antibodies from an ex vivo blood sample. More common methods include latex agglutination, enzyme linked immuno substrate assay, radioimmunoassay, radio immuno sorbent assay, indirect immuno fluorescence and the like. These antibody assays primarily rely on the production of antibody by B-lymphocytes and the measurement of their presence in sufficient quantities in the blood sample.
Serious problems exit however with antibody assays in this type of clinical testing. Firstly, not all disease states, toxins or maladies produce a significant enough antibody response from B-lymphocytes to be useful in antibody assays. A second problem with antibody tests arises when the time for production of antibodies, regardless of the quantity of antibodies produced, is sufficiently long such that by the time a diagnosis is made the patient may have suffered irreparable harm. In some cases several days may need to elapse before measurable quantities of antibodies are present in the blood. In antigen exposure situations where a subjects' medical treatment relies on a quick diagnosis, such low volume or long development time assays are essentially useless. Besides these problems, antibody assays often require highly skilled technicians to perform them and they may need to be performed in a laboratory setting. Not only does that mean the tests are costly, but it means that testing cannot be done on the site of an incident such as a terrorist attack, in the field or a doctors office, since samples must be sent back to the lab.
An alternative to antibody testing is the testing of T-lymphocytes especially memory T-lymphocytes, to determine if there has been a prior exposure to an antigen. T-lymphocytes are known to quickly undergo changes upon second antigen challenge. These changes are collectively referred to as measurement factors. Current lymphocyte testing relies on measurements of morphological factors and/or biochemical factors as an indication of activation by an antigen. Some of the known factors are listed in U.S. Pat. No. 5,480,778. These types of tests also have several difficulties. For example, some require use of radioactive isotopes, samples need to be centrifuged, the time to accomplish the tests can be extremely long (from half a day to several days in length), they require trained personnel to do the test in a lab, require several milliliters of blood, are designed to be used in assays where there is no fluid flow, where samples need to be diluted and the like. Further, the majority of the tests methods do not have the ability to accommodate more than a single mode i.e. a single measurement factor, of analysis. A further problem has been that histochemical staining and cellular morphology are not capable of being done together with most methods. Yet another problem with antibody assays are that T-lymphocytes represent a relatively small portion of leucocytes and even less a percentage of whole undiluted blood making observing or measuring them with small fluid samples extremely difficult.
One method is described in the art that is capable of looking at whole blood and separating it into different cell types. In U.S. Pat. No. 6,350,613 a method of admixing fluorescent dyes with a blood sample is described and target cells can be enumerated by means of a scanning instrument which is able to measure different wavelength of color signals emitted from the target cells in the sample and differentiate target cells from one another by reason of the emitted color signals. This method is described as useful only to separate different cell types in a whole blood sample such as red and while blood cells, doing cell counts and the like. It also describes use in separating populations of leucocyte cells into sub classifications. The patent does not disclose comparing samples of the same cell types for differences in measurement factors, other than quantity of cells information it doesn't discuss any measurement factor observations at all and does not disclose use of this technique to identify cells which have been exposed to an antigen. The patent further does not describe control methods and does not describe use in diagnosis of disease states other than inferring it can perform cell counts.
The apparatus for scanning a fluid sample (for example by fluorescence analysis using computer pixel modeling) is known in the art. Commercial fluorescence readers are readily available and can be programmed by one skilled in the art to read and analyze fluorescence data as needed.
It would be desirable therefore, to have both a device and methods for screening, ex vivo, whole blood or other fluids having leucocytes, for previous exposure to antigens that is easy and safe to use by the person performing the test, is both quantitative and qualitative, can be done in the lab or the field, is accurate, can be done in a relatively short period of time, uses microvolumes (a drop) of blood and wherein the device is disposable.